The aim of the study was using polymerase chain reaction (PCR) technique for detecting the most bacteria causing mastitis in cows as streptococci spp., Escherichia coli and Staphylococcus aureus by using Genus specific primers for each one. One hundred milk samples were collected from cases of subclinical bovine mastitis positive for California Mastitis Test (CMT). The samples were bacteriologically cultivated on to 5% sheep blood agar and also, on different types of media (MacConkey's agar plates, Edward's agar medium and mannitol salt agar) to isolate the major bacteria causing mastitis and identified biochemically. Finally, these bacteria were identified by using PCR technique with a specific primer for each genus. The PCR results revealed that E. coli 16/46 (34.7%), Staphylococcus aureus (S. aureus) 12/40 (30%) and streptococci spp. 11/39 (28.2%). Multiplex PCR for typing of streptococci spp. revealed that St. agalactiae 3/39 (7.69%) and St. dysgalactiae 8/39 (20.51%). In conclusion, in the present study, PCR analysis was used for diagnosis of major bacteria causing subclinical mastitis in cows by using specific primers for each one. The most prevalent bacteria causing subclinical mastitis were E. coli, S. aureus, S. dysgalactia and S. agalactia.